1. Genetic Theory - Overview Pak Sham International Twin Workshop Boulder, 2005

2. The Human Genome 23 Chromosomes, each containing a DNA molecule (Watson and Crick, 1953) 3  10 9 base pairs, completely sequenced (Human Genome Project, 2003) Approximately 24,000 genes, each coding for a polypeptide chain Approximately 10 7 common polymorphisms (variable sites, documented in dbSNP database)

3. Genetic transmission XY Zygote Somatic cells Germ cellsSpermatozoa XX Zygote Somatic cells Germ cellsOva Zygote MitosisMeiosis Fertilization DIPLOID HAPLOIDDIPLOID

4. Sources of Natural Variation Genetic DifferencesEnvironmental Differences Individual Phenotypic Differences

5. Genetic Variation Chromosomal anomalies Insertions / Deletions / Translocations Variable sequence repeats microsatellites (e.g. CACACA….) Single nucleotide polymorphisms (SNPs)

6. Types of Genetic Disease Mendelian diseases e.g. Huntington’s disease, cystic fibrosis A genetic mutation causes the disease Environmental variation usually irrelevant Usually rare Occurs in isolated pedigrees Multifactorial diseases e.g. Coronary heart disease, hypertension, schizophrenia A genetic variant increases the risk of disease Environmental variation usually important Often common Occurs in general population

7. Single-Gene Disorders Human Genome Project completed in 2003 Human Gene Mutation Database contains 44,090 mutations in 1,714 genes Gene Test web site lists genetic tests for 1,093 diseases dbSNP Database Build 123 contains 10,079,771 single nucleotide polymorphisms

8. Autosomal Dominant Disorders

9. AaAaaa AaAa AaAa AaAaAaAa

10. Autosomal Recessive Disorders

11. AaAaAaAa aaAaAaAaAaAA

12. X-linked Dominant Disorders

13. aAaAa AaAaAaaa

14. X-linked Recessive Disorders

15. AAaAa aAAAAaAa

16. Mendelian Segregation

17. Segregation Ratios First discovered by Gregor Mendel in his experiments on the garden pea (published in 1866 and rediscovered in 1900) Form the basis of Mendel’s first law: “law of segregation” Defined as the ratio of affected to normal individuals among the offspring of a particular type of mating.

18. Mendel’s Experiments Pure Lines AAaa F1Aa AA Aa aa 3:1 Segregation Ratio Intercross

19. Mendel’s Experiments Aa aa Aaaa F1Pure line Back cross 1:1 Segregation ratio

20. Segregation Ratios Mode of inheritanceMating typeSegregation ratio Affected:Normal Autosomal dominantAffected x Normal Autosomal recessiveCarrier x Carrier X-linked dominantNormal father x Affected mother X-linked recessiveNormal father x Carrier mother

21. Segregation Ratios Mode of inheritanceMating typeSegregation ratio Affected:Normal Autosomal dominantAffected x Normal1:1 Autosomal recessiveCarrier x Carrier X-linked dominantNormal father x Affected mother X-linked recessiveNormal father x Carrier mother

22. Segregation Ratios Mode of inheritanceMating typeSegregation ratio Affected:Normal Autosomal dominantAffected x Normal1:1 Autosomal recessiveCarrier x Carrier1:3 X-linked dominantNormal father x Affected mother X-linked recessiveNormal father x Carrier mother

23. Segregation Ratios Mode of inheritanceMating typeSegregation ratio Affected:Normal Autosomal dominantAffected x Normal1:1 Autosomal recessiveCarrier x Carrier1:3 X-linked dominantNormal father x Affected mother1:1 X-linked recessiveNormal father x Carrier mother

24. Segregation Ratios Mode of inheritanceMating typeSegregation ratio Affected:Normal Autosomal dominantAffected x Normal1:1 Autosomal recessiveCarrier x Carrier1:3 X-linked dominantNormal father x Affected mother1:1 X-linked recessiveNormal father x Carrier mother1:1 in sons

25. Hardy-Weinberg Law

26. Parental Frequencies GenotypeFrequency AAP AaQ aaR AlleleFrequency AP+Q/2 aR+Q/2

27. Mating Type Frequencies (Random Mating) AAAaaa AAP2P2 PQPR AaPQQ2Q2 QR aaPRQRR2R2

28. Offspring Segregation Ratios AAAaaa AA AA:Aa 0.5:0.5 Aa AA:Aa 0.5:0.5 AA:Aa:aa 0.25:0.5:0.25 Aa:aa 0.5:0.5 aaAaAa:aa 0.5:0.5 aa

29. Offspring Genotype Frequencies GenotypeFrequency AAP 2 +PQ+Q 2 /4 = (P+Q/2) 2 Aa2PR+PQ+QR+Q 2 /2 = 2(P+Q/2)(R+Q/2) aaR2+QR+Q 2 /4 = (R+Q/2) 2

30. Offspring Allele Frequencies AlleleFrequency A(P+Q/2) 2 + (P+Q/2)(R+Q/2) = P+Q/2 a(R+Q/2) 2 + (P+Q/2)(R+Q/2) = R+Q/2

31. Hardy-Weinberg Equilibrium In a large population under random mating: Allele frequencies in the offspring, denoted as p and q, are the same as those in the parental generation. Genotype frequencies in the offspring will follow the ratios p 2 :2pq:q 2, regardless of the genotype frequencies in the parents.

32. Hardy-Weinberg Equilibrium Aa Ap2p2 pqp a q2q2 q pq

33. Hardy-Weinberg Disequilibrium Aa Ap 2 +dpq-dp a q 2 +dq pq

34. Genetic Linkage

35. Genetic Markers Classical Mendelian Disorders Blood groups HLA Antigens Molecular genetic Microsatellites (e.g. CACACA… ) Single-nucleotide polymorphisms (e.g. C/T)

36. High-Throughput Genotyping Extreme multiplexing (multiple markers) DNA Pooling (multiple samples) Maximum throughput of SEQUENOM system at the HKU Genome Research Centre is 100,000 genotypes / day, at a cost of US$ 0.2 per genotype Cost of genotyping set to decrease further – eventually enabling whole-genome association studies to be done.

37. Linkage = Co-segregation A2A4A2A4 A3A4A3A4 A1A3A1A3 A1A2A1A2 A2A3A2A3 A1A2A1A2 A1A4A1A4 A3A4A3A4 A3A2A3A2 Marker allele A 1 cosegregates with dominant disease

38. Crossing-over in meiosis

39. Recombination A1A1 A2A2 Q1Q1 Q2Q2 A1A1 A2A2 Q1Q1 Q2Q2 A1A1 A2A2 Q1Q1 Q2Q2 Likely gametes (Non-recombinants) Unlikely gametes (Recombinants) Parental genotypes

40. Recombination fraction Recombination fraction between two loci = Proportion of gametes that are recombinant with respect to the two loci

41. Double Backcross : Fully Informative Gametes AaBbAaBb aabb AABB aabb AaBbAaBbaabbaabb AabbAabb aaBbaaBb Non-recombinantRecombinant

42. Haplotypes

43. Paternal haplotype Maternal haplotype Genotype

44. Recombination Parental haplotypes Non-recombinantsSingle recombinantsDouble recombinants Possible transmitted haplotypes

45. Linkage Equilibrium Bb Aprpsp aqrqsq rs

46. Linkage Disequilibrium (LD) Bb Apr+dps-dp aqr-dqs+dq rS

47. Decay of LD Gametes 1-   Non-recombinantRecombinant pr+d 1-pr-d pr1-pr ABOthers AB Others Frequency of AB gametes = (1-  )(pr+d)+  pr = pr+(1-  )d

48. Single-Gene Disorders: Some Historical Landmarks 1902: First identified single-gene disorder - alkaptonuria 1956: First identified disease-causing amino acid change: sickle-cell anaemia 1961: First screening program: phenylketonuria 1983: First mapped to chromosomal location: Huntington’s disease 1986: First positionally cloned - chronic granulomatous disease, Duchenne muscular dystrophy 1987: First autosomal recessive disease cloned – cystic fibrosis

49. Types of Genetic Disease Mendelian diseases e.g. Huntington’s disease, cystic fibrosis A genetic mutation causes the disease Environmental variation usually irrelevant Usually rare Occurs in isolated pedigrees Multifactorial diseases e.g. Coronary heart disease, hypertension, schizophrenia A genetic variant increases the risk of disease Environmental variation usually important Often common Occurs in general population

50. Genetic Study Designs

51. Family Studies Case – Control Family Design Compares risk in relatives of case and controls Some terminology Proband Secondary case Lifetime risk / expectancy (morbid risk) Problem: Familial aggregation can be due to shared family environment as well as shared genes

52. Family Studies: Schizophrenia Relationship to ProbandLifetime Risk of Schizophrenia (%) Unrelated1 First cousins2 Uncles/Aunts2 Nephews/Nieces4 Grandchildren5 Half siblings6 Parents6 Siblings9 Children13 From: Psychiatric Genetics and Genomics. MuGuffin, Owen & Gottesman, 2002

53. Twin Studies Studies risk of disease (concordance rates) in cotwins of affected MZ and DZ Twin Under the equal environment assumption, higher MZ than DZ concordance rate implies genetic factors Problems: Validity of equal environment assumption Generalizability of twins to singletons

54. Twin Studies: Schizophrenia ZygosityConcordance (%) Dizygotic (DZ)17 Monozygotic (MZ)48 From: Psychiatric Genetics and Genomics. MuGuffin, Owen & Gottesman, 2002

55. Adoption Studies Adoptees’ method compares Adoptees with an affected parent Adoptees with normal parents Adoptee’s family method compares Biological relatives of adoptees Adoptive relatives of adoptees Problems: Adoption correlated with ill-health/psychopathology in parents Adoptive parents often rigorously screened

56. Adoption Studies: Schizophrenia Adoptees ofRisk of Schizophrenia (%) Schizophrenic parents8 Control parents2 From: Finnish Adoption Study, as summarised in Psychiatric Genetics and Genomics. MuGuffin, Owen & Gottesman, 2002

57. Biometrical Genetic Model

58. 0 d a-a Trait Means AA Aa aa a d -a aaAaAA Fisher’s convention: the midpoint between the trait means of the two homozygous genotypes is designated as 0

59. Biometrical Model: Mean GenotypeAAAaaa Frequencyp 2 2pqq 2 Trait (x)ad-a Mean m = p 2 (a) + 2pq(d) + q 2 (-a) = (p-q)a + 2pqd

60. Biometrical Model: Variance GenotypeAAAaaa Frequencyp 2 2pqq 2 (x-m) 2 (a-m) 2 (d-m) 2 (-a-m) 2 Variance = (a-m) 2 p 2 + (d-m) 2 2pq + (-a-m) 2 q 2 = 2pq[a+(q-p)d] 2 + (2pqd) 2 = V A + V D

61. Average Allelic Effect Effect of gene substitution: a  A If background allele is a, then effect is (a+d) If background allele is A, then effect is (a-d) Average effect of gene substitution is therefore  = q(a+d) + p(a-d) = a + (q-p)d Additive genetic variance is therefore V A = 2pq  2

62. Additive and Dominance Variance aaAaAA m -a a d  

63. Cross-Products of Deviations for Pairs of Relatives AAAaaa AA(a-m) 2 Aa(a-m)(d-m)(d-m) 2 aa(a-m)(-a-m)(-a-m)(d-m)(-a-m) 2 The covariance between relatives of a certain class is the weighted average of these cross-products, where each cross-product is weighted by its frequency in that class.

64. Covariance of MZ Twins AAAa aa AAp 2 Aa02pq aa00q 2 Covariance = (a-m) 2 p 2 + (d-m) 2 2pq + (-a-m) 2 q 2 = 2pq[a+(q-p)d] 2 + (2pqd) 2 = V A + V D

65. Covariance for Parent-offspring (P-O) AAAaaa AAp 3 Aap 2 qpq aa0 pq 2 q 3 Covariance = (a-m) 2 p 3 + (d-m) 2 pq + (-a-m) 2 q 3 + (a-m)(d-m)2p 2 q + (-a-m)(d-m)2pq 2 = pq[a+(q-p)d] 2 = V A / 2

66. Covariance for Unrelated Pairs (U) AAAaaa AAp 4 Aa2p 3 q4p 2 q 2 aap 2 q 2 2pq 3 q 4 Covariance = (a-m) 2 p 4 + (d-m) 2 4p 2 q 2 + (-a-m) 2 q 4 + (a-m)(d-m)4p 3 q + (-a-m)(d-m)4pq 3 + (a-m)(-a-m)2p 2 q 2 = 0

67. Covariance for DZ twins Genotype frequencies are weighted averages: ¼ MZ twins (when  =1) ½ Parent-offspring (when  =1) ¼ Unrelated (when  =0) Covariance = ¼(V A +V D ) + ½(V A /2) + ¼ (0) = ½V A + ¼V D

68. Covariance: General Relative Pair Covariance= Prob(  =1)(V A +V D ) + Prob(  =1/2)(V A /2) + Prob(  =0)(0) = (Prob(  =1)+Prob(IBD=1/2)/2)V A + Prob(  =1) V D = E(  )V A + Prob(  =1)V D = 2  V A +  V D

69. Total Genetic Variance Heritability is the combined effect of all loci total component = sum of individual loci components V A = V A1 + V A2 + … + V AN V D = V D1 + V D2 + … + V DN CorrelationsMZDZP-OU V A (2  )10.50.50 V D (  ) 10.2500

70. Quantitative Genetics

71. Examples of quantitative traits Blood Pressure (BP) Body Mass Index (BMI) Blood Cholesterol Level General Intelligence (G) Many quantitative traits are relevant to health and disease

72. Quantitative Traits 1 Gene  3 Genotypes  3 Phenotypes 2 Genes  9 Genotypes  5 Phenotypes 3 Genes  27 Genotypes  7 Phenotypes 4 Genes  81 Genotypes  9 Phenotypes Central Limit Theorem  Normal Distribution

73. Continuous Variation   +1.96   -1.96  2.5% 95% probability Normal distribution Mean , variance  2

74. Bivariate normal

75. Familial Covariation Relative 1 Relative 2 Bivariate normal disttribution

76. Correlation due to Shared Factors Francis Galton: Two Journeys starting at same time Denmark HillVictoria Paddington Brixton A B C Journey Times: A+B and A+C Shared ACovariance Correlation

77. Shared Genes ABCD ACACADAD Gene A is shared: = Identity-By-Descent (IBD)  Shared Phenotypic Effects At any chromosomal location, two individuals can share 0, 1 or 2 alleles.

78. Identity by Descent (IBD) Two alleles are IBD if they are descended from and replicates of the same ancestral allele 7 AaAa AaAa AaAa A aa AA Aa 1 2 3456 8

79. IBD: Parent-Offspring ABABCDCD ACAC If the parents are unrelated, then parent-offspring pairs always share 1 allele IBD

80. IBD: MZ Twins ABABCDCD ACAC MZ twins always share 2 alleles IBD ACAC

81. IBD: Half Sibs ABABCDCD ACAC EE CE/DE IBD SharingProbability 0½ 1½

82. IBD: Full Sibs 01 12 IBD of paternal alleles IBD of maternal alleles 01 0 1

83. IBD: Full Sibs IBD SharingProbability 01/4 11/2 21/4 Average IBD sharing = 1

84. Genetic Relationships  (kinship coefficient): Probability of IBD between two alleles drawn at random, one from each individual, at the same locus.  : Probability that both alleles at the same locus are IBD Relationship   MZ twins0.51 Parent-offspring0.250 Full sibs0.250.25 Half sibs0.1250

85. Proportion of Alleles IBD (  ) Relatiobship  E(  )Var(  ) MZ0.5 1 0 Parent-Offspring0.250.5 0 Full sibs0.250.50.125 Half sibs0.1250.250.0625 Most relationships demonstrate variation in  across the chromosomes Proportion of alleles IBD = Number of alleles IBD / 2

86. Classical Twin Analysis MZ TwinsDZ Twins Average genetic sharing 100%50% Phenotypic correlation = >   Genetic influences No genetic influences Note: Equal Environment Assumption

87. P1P1 A1A1 C1C1 E1E1 P2P2 A2A2 C2C2 E2E2 1 [0.5/1] ea c e c a ACE Model for twin data

88. Implied covariance matrices  Difference between MZ and DZ covariance ~ Genetic Variance / 2

89. Heritability Is proportion of phenotypic variance due to genetic factors Is population-specific May change with changes in the environment A high heritability does not preclude effective prevention or intervention Most human traits have heritability of 30% – 90%

90. Liability-Threshold Models

91. Single Major Locus (SML) Model GenotypePhenotype f2f2 AA aa Aa Disease Normal f1f1 f0f0 1- f 2 1- f 1 1- f 0 “Penetrance parameters”

92. Liability-Threshold Model

93. Liability-threshold model General population Relatives of probands

94. Threshold Model with SML f(X)f(X) X AA Aa aa

95. Quantitative Trait Linkage

96. QTL Linkage Analysis Local genetic sharing 21 Phenotypic correlation Linkage No linkage 0 DZ Twins / Sibling Pairs

97. P1P1 Q1Q1 A1A1 E1E1 P2P2 Q2Q2 A2A2 E2E2 0.5 eqaeaq QTL linkage model for sib pairs

98. Exercise From the path diagram write down the implied covariance matrices for sib pairs with proportion IBD sharing of 0, 0.5 and 1.

99. Quantitative Association

100. Allelic Association disease susceptibility allele is more frequent in cases than in controls ControlsCases Example: Apolipoprotein E  4 allele increases susceptibility to Alzheimer’s disease

101. Analysis of Means AAAaaa No association Association Phenotype Genotype

102. Causes of association Direct: allele increases risk of disease Indirect: allele associated with a risk- increasing allele through tight linkage “Spurious”: allele associated with disease through confounding variable (e.g. population substructure).

103. Haplotype association Mutational event on ancestral chromosome Multiple generations Present mutation- bearing chromosomes with variable preserved region

104. Complex Disorders: Some Historical Landmarks 1875: Use of twins to disentangle nature from nurture (Galton) 1918: Polygenic model proposed to reconcile quantitative and Mendelian genetics (Fisher) 1965: Liability-threshold model postulated for common congenital malformations (Carter) 1960’s: Association between blood groups and HLA antigens with disease 1990’s: Identification of APOE-e4 as a susceptibility allele for dementia 2000’s: International HapMap Project

105. of the Behavior Genetic Association.